A fluorescence dye that provides an internal reference to which the reporter dye signal can be normalized during data analysis by the software. This type of normalization is necessary to correct for fluctuations from well to well caused by changes in concentration or volume. ROX is the most commonly used passive reference dye. After the run, in the multicomponent view, the passive reference dye (if used) should have lower fluorescence than the reporter dyes. Although its signal level should remain constant throughout the experiment, a dip in the ROX signal levels in late cycles (>35 cycles) is nothing to worry about. Not all instruments require the use of a passive reference dye in reaction setup. In those that do not require passive reference dye (like Stratagene and Bio-Rad), the master mix should not contain too much ROX (around 30nM final concentration is allowed). For the effect of ROX concentration on threshold, see ABI Publication: Understanding Ct Value.